Areej Abuhammad | University of Jordan (original) (raw)
Uploads
Papers by Areej Abuhammad
Acta crystallographica. Section D, Biological crystallography, 2013
Arylamine N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) plays an important role in ... more Arylamine N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) plays an important role in the intracellular survival of the microorganism inside macrophages. Medicinal chemistry efforts to optimize inhibitors of the TBNAT enzyme have been hampered by the lack of a three-dimensional structure of the enzyme. In this paper, the first structure of TBNAT, determined using a lone crystal produced using crossseeding with the homologous protein from M. marinum, is reported. Despite the similarity between the two enzymes (74% sequence identity), they show distinct physical and biochemical characteristics. The structure elegantly reveals the characteristic features of the protein surface as well as details of the active site of TBNAT relevant to drug-discovery efforts. The crystallographic analysis of the diffraction data presented many challenges, since the crystal was twinned and the habit possessed pseudo-translational symmetry.
Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of... more Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of tuberculosis (TB). Cholesterol plays a critical role in the persistence of M. tuberculosis within the macrophage during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine Nacetyltransferase (NAT) is encoded within a gene cluster that is involved in the cholesterol sterol-ring degradation and is essential for intracellular survival. The ability of the NAT from M. tuberculosis (TBNAT) to utilise propionyl-CoA links it to the cholesterol-catabolism pathway. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. TBNAT has been investigated as a potential target for TB therapies. From a previous high-throughput screen, 3-benzoyl-4-phenyl-1-methylpiperidinol was identified as a selective inhibitor of prokaryotic NAT that exhibited antimycobacterial activity. The compound resulted in time-dependent irreversible inhibition of the NAT activity when tested against NAT from M. marinum (MMNAT). To further evaluate the antimycobacterial activity and the NAT inhibition of this compound, four piperidinol analogues were tested. All five compounds exert potent antimycobacterial activity against M. tuberculosis with MIC values of 2.3-16.9 mM. Treatment of the MMNAT enzyme with this set of inhibitors resulted in an irreversible time-dependent inhibition of NAT activity. Here we investigate the mechanism of NAT inhibition by studying protein-ligand interactions using mass spectrometry in combination with enzyme analysis and structure determination. We propose a covalent mechanism of NAT inhibition that involves the formation of a reactive intermediate and selective cysteine residue modification. These piperidinols present a unique class of antimycobacterial compounds that have a novel mode of action different from known anti-tubercular drugs.
Protein Expression and …, Jan 1, 2011
Arylamine N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) has been proposed as a drug... more Arylamine N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) has been proposed as a drug target for latent tuberculosis treatment. The enzyme is essential for the survival of the mycobacterium in macrophages. However, TBNAT has been very difficult to generate as a soluble protein. In this work we describe production of soluble recombinant TBNAT at a reasonable yield achieved by subcloning the tbnat gene with a purification His-tag into the pVLT31 plasmid, and subsequent optimisation of the induction conditions. The expression system results in soluble protein optimised upon extended (60 h) low level isopropyl b-D-1-thiogalactopyranoside level induction (100 lM) at a temperature of 15°C. The level of TBNAT expression obtained in E. coli has been significantly improved from $2 mg to a final yield of up to 16 mg per litre of culture at a purity level suitable for structural studies. The molecular mass of 31310 Da was confirmed using mass spectroscopy and the oligomerisation state was determined. The stability of TBNAT in different buffer systems was investigated by thermal shift assays and sufficient protein is now available for the screening of chemical libraries for inhibitors.
The Protein …, Jan 1, 2009
… Section F: Structural …, Jan 1, 2009
![Research paper thumbnail of Analysis of [beta]-amino alcohols as inhibitors of the potential anti-tubercular target N-acetyltransferase](https://attachments.academia-assets.com/6181167/thumbnails/1.jpg)
](Bioorganic & Medicinal …, Jan 1, 2010
Protein & Cell, Jan 1, 2010
Journal of Molecular Graphics and …, Jan 1, 2007
In this project, several docking conditions, scoring functions and corresponding protein-aligned ... more In this project, several docking conditions, scoring functions and corresponding protein-aligned molecular field analysis (CoMFA) models were evaluated for a diverse set of neuraminidase (NA) inhibitors. To this end, a group of inhibitors were docked into the active site of NA. The docked structures were utilized to construct a corresponding protein-aligned CoMFA models by employing probe-based (H+, OH, CH3) energy grids and genetic partial least squares (G/PLS) statistical analysis. A total of 16 different docking configurations were evaluated, of which some succeeded in producing self-consistent and predictive CoMFA models. However, the best model coincided with docking the ionized ligands into the hydrated form of the binding site via PLP1 scoring function (r 2 LOO ¼ 0:735, r 2 PRESS against 24 test compounds = 0.828). The highest-ranking CoMFA models were employed to probe NA-ligand interactions. Further validation by comparison with a co-crystallized ligand-NA crystallographic structure was performed. This combination of docking/scoring/CoMFA modeling provided interesting insights into the binding of different NA inhibitors. #
Journal of chemical information …, Jan 1, 2009
Neuraminidase (NA) enzyme is one of the valid targets against influenza viruses. With this in min... more Neuraminidase (NA) enzyme is one of the valid targets against influenza viruses. With this in mind, the pharmacophoric space of influenza NA was explored using three sets of diverse inhibitors. Subsequently, genetic algorithm and multiple linear regression analysis were employed to select optimal combinations of pharmacophoric models and 2D descriptors capable of yielding self-consistent and predictive quantitative structure-activity relationships (QSARs) against 181 training compounds. The optimal QSAR equations were validated against 43 external test compounds with r 2 PRESS values ranging from 0.488 to 0.591. Interestingly, five orthogonal pharmacophores emerged in the optimal QSAR equations suggesting the existence of several distinct ligand/NA binding modes within the NA binding pocket. The resulting pharmacophores were complemented with tight shape constraints and employed as three-dimensional (3D) search queries against the National Cancer Institute (NCI) list of compounds. Several hits exhibited potent inhibitory activities against NA. The highest ranking hit demonstrated an in Vitro IC 50 value of 1.8 µM. Docking studies supported the binding modes suggested by our pharmacophore/QSAR analysis.
European journal of …, Jan 1, 2009
Homology modeling is becoming a valid method for obtaining three-dimensional coordinates for prot... more Homology modeling is becoming a valid method for obtaining three-dimensional coordinates for proteins. However, it is hard to judge the qualities of the resulting models warranting robust subsequent validations. In an attempt to evaluate the quality of Melanin-concentrating hormone 1 receptor (MCH1R) homology models, a number of homology structures were scanned for potential binding cavities. Subsequently, a group of 35 benzylpiperidines' MCH1R inhibitors were docked into each of the proposed binding sites via four different scoring functions. The docked structures were utilized to construct corresponding protein-aligned comparative molecular field analysis (CoMFA) models by employing probebased (H þ , OH, CH 3 ) energy grids and genetic partial least squares (G/PLS) statistical analysis. The docking-based alignment succeeded in accessing self-consistent CoMFA models upon employing JAIN scoring function in one of the proposed binding pockets in a particular homology model. Furthermore, a ligand-based pharmacophore model was developed for the same set of inhibitors and was found to agree with the successful docking configuration. Therefore, we proved that the overall procedure of docking, scoring, and CoMFA evaluation can be a useful tool to validate homology models, which can be of value for structure-based design, in-silico screening, and in understanding the structural basis of ligand binding to MCH1R.
Teaching Documents by Areej Abuhammad
Acta crystallographica. Section D, Biological crystallography, 2013
Arylamine N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) plays an important role in ... more Arylamine N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) plays an important role in the intracellular survival of the microorganism inside macrophages. Medicinal chemistry efforts to optimize inhibitors of the TBNAT enzyme have been hampered by the lack of a three-dimensional structure of the enzyme. In this paper, the first structure of TBNAT, determined using a lone crystal produced using crossseeding with the homologous protein from M. marinum, is reported. Despite the similarity between the two enzymes (74% sequence identity), they show distinct physical and biochemical characteristics. The structure elegantly reveals the characteristic features of the protein surface as well as details of the active site of TBNAT relevant to drug-discovery efforts. The crystallographic analysis of the diffraction data presented many challenges, since the crystal was twinned and the habit possessed pseudo-translational symmetry.
Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of... more Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of tuberculosis (TB). Cholesterol plays a critical role in the persistence of M. tuberculosis within the macrophage during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine Nacetyltransferase (NAT) is encoded within a gene cluster that is involved in the cholesterol sterol-ring degradation and is essential for intracellular survival. The ability of the NAT from M. tuberculosis (TBNAT) to utilise propionyl-CoA links it to the cholesterol-catabolism pathway. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. TBNAT has been investigated as a potential target for TB therapies. From a previous high-throughput screen, 3-benzoyl-4-phenyl-1-methylpiperidinol was identified as a selective inhibitor of prokaryotic NAT that exhibited antimycobacterial activity. The compound resulted in time-dependent irreversible inhibition of the NAT activity when tested against NAT from M. marinum (MMNAT). To further evaluate the antimycobacterial activity and the NAT inhibition of this compound, four piperidinol analogues were tested. All five compounds exert potent antimycobacterial activity against M. tuberculosis with MIC values of 2.3-16.9 mM. Treatment of the MMNAT enzyme with this set of inhibitors resulted in an irreversible time-dependent inhibition of NAT activity. Here we investigate the mechanism of NAT inhibition by studying protein-ligand interactions using mass spectrometry in combination with enzyme analysis and structure determination. We propose a covalent mechanism of NAT inhibition that involves the formation of a reactive intermediate and selective cysteine residue modification. These piperidinols present a unique class of antimycobacterial compounds that have a novel mode of action different from known anti-tubercular drugs.
Protein Expression and …, Jan 1, 2011
Arylamine N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) has been proposed as a drug... more Arylamine N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) has been proposed as a drug target for latent tuberculosis treatment. The enzyme is essential for the survival of the mycobacterium in macrophages. However, TBNAT has been very difficult to generate as a soluble protein. In this work we describe production of soluble recombinant TBNAT at a reasonable yield achieved by subcloning the tbnat gene with a purification His-tag into the pVLT31 plasmid, and subsequent optimisation of the induction conditions. The expression system results in soluble protein optimised upon extended (60 h) low level isopropyl b-D-1-thiogalactopyranoside level induction (100 lM) at a temperature of 15°C. The level of TBNAT expression obtained in E. coli has been significantly improved from $2 mg to a final yield of up to 16 mg per litre of culture at a purity level suitable for structural studies. The molecular mass of 31310 Da was confirmed using mass spectroscopy and the oligomerisation state was determined. The stability of TBNAT in different buffer systems was investigated by thermal shift assays and sufficient protein is now available for the screening of chemical libraries for inhibitors.
The Protein …, Jan 1, 2009
… Section F: Structural …, Jan 1, 2009
Bioorganic & Medicinal …, Jan 1, 2010
Protein & Cell, Jan 1, 2010
Journal of Molecular Graphics and …, Jan 1, 2007
In this project, several docking conditions, scoring functions and corresponding protein-aligned ... more In this project, several docking conditions, scoring functions and corresponding protein-aligned molecular field analysis (CoMFA) models were evaluated for a diverse set of neuraminidase (NA) inhibitors. To this end, a group of inhibitors were docked into the active site of NA. The docked structures were utilized to construct a corresponding protein-aligned CoMFA models by employing probe-based (H+, OH, CH3) energy grids and genetic partial least squares (G/PLS) statistical analysis. A total of 16 different docking configurations were evaluated, of which some succeeded in producing self-consistent and predictive CoMFA models. However, the best model coincided with docking the ionized ligands into the hydrated form of the binding site via PLP1 scoring function (r 2 LOO ¼ 0:735, r 2 PRESS against 24 test compounds = 0.828). The highest-ranking CoMFA models were employed to probe NA-ligand interactions. Further validation by comparison with a co-crystallized ligand-NA crystallographic structure was performed. This combination of docking/scoring/CoMFA modeling provided interesting insights into the binding of different NA inhibitors. #
Journal of chemical information …, Jan 1, 2009
Neuraminidase (NA) enzyme is one of the valid targets against influenza viruses. With this in min... more Neuraminidase (NA) enzyme is one of the valid targets against influenza viruses. With this in mind, the pharmacophoric space of influenza NA was explored using three sets of diverse inhibitors. Subsequently, genetic algorithm and multiple linear regression analysis were employed to select optimal combinations of pharmacophoric models and 2D descriptors capable of yielding self-consistent and predictive quantitative structure-activity relationships (QSARs) against 181 training compounds. The optimal QSAR equations were validated against 43 external test compounds with r 2 PRESS values ranging from 0.488 to 0.591. Interestingly, five orthogonal pharmacophores emerged in the optimal QSAR equations suggesting the existence of several distinct ligand/NA binding modes within the NA binding pocket. The resulting pharmacophores were complemented with tight shape constraints and employed as three-dimensional (3D) search queries against the National Cancer Institute (NCI) list of compounds. Several hits exhibited potent inhibitory activities against NA. The highest ranking hit demonstrated an in Vitro IC 50 value of 1.8 µM. Docking studies supported the binding modes suggested by our pharmacophore/QSAR analysis.
European journal of …, Jan 1, 2009
Homology modeling is becoming a valid method for obtaining three-dimensional coordinates for prot... more Homology modeling is becoming a valid method for obtaining three-dimensional coordinates for proteins. However, it is hard to judge the qualities of the resulting models warranting robust subsequent validations. In an attempt to evaluate the quality of Melanin-concentrating hormone 1 receptor (MCH1R) homology models, a number of homology structures were scanned for potential binding cavities. Subsequently, a group of 35 benzylpiperidines' MCH1R inhibitors were docked into each of the proposed binding sites via four different scoring functions. The docked structures were utilized to construct corresponding protein-aligned comparative molecular field analysis (CoMFA) models by employing probebased (H þ , OH, CH 3 ) energy grids and genetic partial least squares (G/PLS) statistical analysis. The docking-based alignment succeeded in accessing self-consistent CoMFA models upon employing JAIN scoring function in one of the proposed binding pockets in a particular homology model. Furthermore, a ligand-based pharmacophore model was developed for the same set of inhibitors and was found to agree with the successful docking configuration. Therefore, we proved that the overall procedure of docking, scoring, and CoMFA evaluation can be a useful tool to validate homology models, which can be of value for structure-based design, in-silico screening, and in understanding the structural basis of ligand binding to MCH1R.